Project Summary Sexually transmitted infections (STIs) are a major public health challenge and a serious women's health issue, as women can suffer severe complications from these infections: pelvic inflammatory disease (PID), infertility, and predisposition to life-threatening ectopic pregnancy. However, the majority of these infections in the female reproductive tract (FRT) are asymptomatic. How infection in the FRT causes such a wide range of clinical outcomes in the absence of symptoms remains unknown. The primary obstacle to understanding STIs of the FRT is the lack of a model that reasonably mimics all aspects of human infection. The cervix is the initiation site for STIs in the FRT. The cervical mucosa is not uniform, composed of multilayered non-polarized squamous epithelial cells at the ectocervix, a single layer of polarized columnar cells at the endocervix, and the progressively changing epithelia in the transformation zone. While tissue culture models have contributed significantly to explaining specific host-pathogen interactions, how STI pathogens deal with different epithelia for infection is unclear, as no cell culture model can mimic the varying mucosal surfaces of the human cervix. To overcome these obstacles, we are developing a new infection model using human cervical tissue explants to address our long-term goal: to delineate the mechanisms by which STI pathogens infect the FRT. To pursue this goal, this proposal focuses on the cellular mechanism by which Neisseria gonorrhoeae (GC) modulates the infection process in the human cervix. GC causes gonorrhea that is the second most common STI and a public health crisis worldwide due to the upsurge of multi-drug resistant GC. The surface molecules of GC undergo phase variation, which has been implicated in its broad infection outcomes. We hypothesize that the expression of pili and distinct variants of opacity associated proteins (Opa) allows for changes in GC infectivity, while the properties of epithelial cells of the human cervix determine which regions are vulnerable to GC infections. To test the hypothesis, we will use our human cervical tissue model and isogenic strains of GC that express invariable Opas and pili to define the cellular mechanism by which pili and Opa phase variation and the distinct properties of cervical epithelial cells regulate GC infection in the FRT. Our cervical explant model breaks a major barrier of the field, making it possible for the first time to examine cellular events occurring during GC infection to their in vivo targeted epithelial cells under a physiologically relevant environment. These studies will reveal new mechanisms that can finally explain how GC manipulate signaling and cytoskeleton based on their surface molecules and the type of epithelial cells with which they interact to switch the cervical infection between colonizing and penetrating nature. The new tissue model and infection mechanisms established by this project may fundamentally change our way to pursue the understanding of STIs and open new avenues for interventive drug designs for the prevention of STIs.